Pharmaceutical composition comprising chito-oligomers

ABSTRACT

Compositions are provided comprising chito-oligomers obtainable from chitin, comprising oligomers of N-acetyl glucosamine (NAG) and glucosamine, wherein at least 50% of the oligomers have a chain length of about 2-50, and the degree of deacetylation of the oligomers is in the range of about 0-70%, preferably about 30-50%. The compositions are highly useful as pharmaceutical compositions for treatment of joint disorders such as rheumatoid arthritis and osteoarthritis. Also provided are methods for treatment of joint disorders and treatment against inflammatory activity.

FIELD OF INVENTION

The current invention is within the pharmaceutical field, specificallyfor treatment of joint disorders such as rheumatoid arthritis andosteoarthritis.

TECHNICAL BACKGROUND

Chitin and chitosan are biopolymers, which typically are obtained fromcrustacean shell wastes, but can also be obtained from certain fungi.Chitosan may be prepared from chitin by chemical deacetylation. This istypically achieved by hydrolysing the N-acetyl linkage in chitin withconcentrated alkali (40-50% NaOH or KOH). By definition, chitosan isgenerally described as a copolymer of D-glucosamine (GlcN) andN-acetyl-D-glucosamine (GlcNAc or NAG), which is insoluble in water atpH above 6.2—the isoelectric point of the free amine group—but dissolvesat pH below 6.2 (See Scheme 1 and 2). In chitosan, 65-100% of themonomer units are D-glucosamine, which is usually described as 65-100%deacetylated chitin. Chemical and biological properties of chitosan aredirectly influenced by the degree of deacetylation (DDA) and degree ofpolymerisation (DP), i.e. the chain length of the polymer.

In solution at pH below 6.2, and when amine groups of the D-glucosamineresidues are protonated, chitosan is a positively charged polymer. Beingan amine, chitosan is a weak base and can form salts with acids, such ascarboxylic and mineral acids. Most of these salts are water-soluble.

In its natural form, chitin is insoluble in water. However, it can bemade water-soluble by partial deacetylation through alkali treatment[1]. Partially deacetylated chitin with DDA of 35-50% is soluble inwater at a wide range of pH. This form of water-soluble chitin has beenshown to be an excellent substrate for chitin converting enzymes [2, 3].Moreover, preparation of water soluble chitin has shown to be anecessary step to retain high yield of chito-oligomers using chitinases,since insoluble chitin is hydrolyzed very slowly by chitinases [1].

Chito-oligomers (COs) and low molecular weight chitin and chitosan areshorter segments, made from the higher molecular weight polysaccharidesby hydrolysis of the beta-(1,4)-bonds that link the monomers.Chito-oligomers refer herein to short to medium-length polymers,preferably having a degree of polymerization (DP) in the range of 2 to50, corresponding to a molecular mass of about 360 to about 10.000 Da.COs made from water-soluble chitin (DDA 35-50%) maintain their watersolubility. COs are made either chemically by using strong acids such ashydrochloric acid, catalysing the hydrolysis of the beta-(1,4)-bond athigh temperatures, or by using enzymatic hydrolysis [4,5]. Enzymatichydrolysis is favored since the process is easier to control andconditions are much milder and involve less risk of side reactionsresulting in chemical modifications of the material.

Arthritis is a general term for inflammation of the joint(s), andsometime used to include all joint disorders. Osteoarthritis is thecommonest form of joint disease in which there is damage to the surfaceof the joint and an abnormal reaction in the underlying bone. Otherterms are used to describe this disease, such as ‘osteoarthrosis’,‘arthrosis’ and ‘degenerative joint disease’. The disease mainly affectsknees, hips, and hands (most common), as well as the foot and the neckand back. Rheumatoid arthritis is a common inflammatory disease of thejoints, which causes inflammation of the lining membrane of the joint(synovium). This results in more swelling and other signs ofinflammation than is usual in osteoarthritis, and can lead to severedamage of the joints.

Bioactivity of Chito-oligosaccarides:

Biological activity of chitin and chitosan is abundantly documented inthe literature. Bioactivity studies have clearly demonstrated theimportance of degree of polymerization (DP) as well as degree ofdeacetylation (DDA) [6]. In plants, the oligomers of DP 5-7 are moreactive than DP 1-4 [7]. The reason has been related to the ability ofso-called chitinase-like proteins (CLPs) to bind chito-oligomers. Theseproteins share a high sequence homology and a structural relationshipwith family 18 chitinases [8]. The CLPs lack catalytic activity becauseof a single point mutation in their catalytic domain but they maintaintheir oligosaccharide binding ability, which usually involves 5-7chito-oligosaccharide units.

N-Acetyl-Glucosamine, Chito-oligosaccharides and Hyaluronan: Glucosamine(GN or GlcN) is a modified glucose with NH₂ replacing the OH group onthe carbon two in the sugar molecule. In animal cells, glucosamine isonly found in two forms; as glucosamine-6-phosphate (GN-6-P) andN-acetylglucosamine (NAG or GlcNAc). The amino sugar GN-6-P issynthesized from glutamine and fructose-6-phosphate (F-6-P). Thisreaction is catalysed by glucosamine synthase and is the rate limitingstep in amino sugar biosynthesis. GN-6-P is the precursor to allhexosamines and hexosamine derivatives. GN-6-P can subsequently beacetylated by acetyl coenzyme A to N-acetyl glucosamine (NAG). NAG cansubsequently be converted into N-acetyl galactosamine or N-acetylmannosamine. These three amino sugars are important in glycosylation ofproteins as well as building blocks for glycolipids, glycosaminoglycans(GAG), hyaluronan and proteoglycans. Hyaluronan (HA), the backbone ofmany proteoglycans, is a polysaccharide (up to 25,000 sugar units)composed of repeating disaccharide units of NAG and glucuronic acid(GlcA). HA is thought to be the earliest evolutionary form of GAG. HA isnot only an important polysaccharide in cartilage, synovial fluid,viterous humor of the eye and in the skin of vertebrates, but may alsoplay an important role in tissue organization, morphogenesis, cancermetastasis, wound healing and inflammation [9]. It is produced in largequantities during wound repair, and is an essential constituent of jointfluid (synovial fluid), where it serves as a lubricant [10]. NAGincreases the synthesis of hyaluronan by mesothelial cells andfibroblasts in a dose-dependent manner [11]. HA is secreted from cellsby an enzyme complex, named HA synthases (HAS) which is embedded in theplasma membrane [9]. These enzymes are thought to have evolved fromchitin synthases or cellulose synthases [9]. A mouse HA synthase (HAS1)is capable to synthesize HA in vitro, when it is supplied with UDP-GlcAand UDP-NAG [12]. When HAS1 is incubated with UDP-NAG alone, itsynthesizes chito-oligosaccarides (COs) [12]. A demonstration of similaractivity of eukariotic HA synthases in vivo, would suggest novelfunctions for COs in mammals [9]. COs are produced in vivo during thedevelopment of vertebrates (Xenopus, zebrafish and mouse), where thechitinase-like DG42/HAS subfamily synthesizes both COs and HA duringcell differentiation and the COs have been shown to be vital for anormal anterior/posterior axis formation in the late gastrla [9, 12-16],reviewed by [8].

Recent studies have suggested methods of treating arthritis byadministration of glucosamine. These studies have shown thatadministration of glucosamine tends to normalize cartilage metabolism,inhibiting degradation, and stimulating the synthesis of proteoglycans,resulting in aortial restoration of the articular function. Thetherapeutic efficacy of treatment with glucosamine has been demonstratedin a number of animal and human studies.

U.S. Pat. No. 6,117,851 [17] teaches that (poly)-N-acetyl glucosamine(poly-NAG), i.e. chitin can be used to treat osteoarthritis and/oralleviate symptoms thereof. However, chitin, acting like insoluble fibrein the gut, is unlikely to be digested and absorbed. Also, due to itspoor solubility in the gut environment, chitin is not likely to beefficiently hydrolysed by the recently discovered acidic mammalianchitinase (AMCase) [18] or intestinal bacteria producing lower molecularchitin fragments available for absorption. Partially deacetylated chitinhowever, is water-soluble at any pH and readily available as substratefor AMCase or intestinal flora.

Chito-oligomer Activity on Immune Response and Inflammatory Reactions—Chondrocytes and Macrophages:

Chitin and chitosan have been suggested to possess immunostimulatingactivity in mammals [19-22]. Also, chitin and chitosan have been studiedin wound healing and artificial skin substitutes for some years [19-22].In these studies, chitin and chitosan have showed a significantinhibitory effect on nitric oxide (NO) production by activatedmacrophages. Hexa-N-acetylchitohexaose (GlcNAc)₆ andpenta-N-acetylchitopentaose (GlcNAc)₅ also inhibited NO production butwith less potency. These results indicate that the positive effect ofchitinous materials on wound healing is at least partly related to theinhibition of NO production by the activated macrophages [23]. It hasalso been shown that both glucosamine and N-acetylglucosamine inhibit NOproduction in normal human articular chondrocytes and thatN-acetylglucosamine has a novel mechanism for the inhibition ofinflammatory processes [24].

The chitinase-like protein YKL-40, also called human cartilageglycoprotein-39 (HC gp-39), is a member of family 18 chitinases [25].YKL-40 is secreted by chondrocytes, synovial cells, and macrophages[26]. HC gp-39 (YKL-40) appears to be induced in aging human and youngosteroarthritis patients [28]. It has been reported that YKL-40 has arole as an auto-antigen in rheumatoid arthritis (RA) [29-31] and it isexpressed in diseased human osteoarthritic cartilage and osteophyte, butnot in non-diseased tissue [32].

REFERENCES

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SUMMARY OF INVENTION

We have now found that compositions comprising chito-oligomers (2- to50-mers) provide remarkably good results in alleviating the symptoms ofjoint disorders such as arthritis. Results indicate that thechito-oligomers appear to be surprisingly more effective than monomerglucosamine in this regard, particularly as administration of theoligomers substantially reduces inflammations. We suggest that chitinousligands, i.e. chito-oligosaccharides (COs), by binding to the YKL-40 orsimilar chitinase like proteins, might work to reduce their expressionand/or reduce auto-antigen activity by masking or changing theirepitopes.

We have observed anti-inflammatory effects in human subjects sufferingfrom rheumatoid arthritis after administration of chito-oligomers for3-4 weeks. These anti-inflammatory effects could possibly influencechondrocytes, macrophages and possibly osteoblasts via YKL-40.

Preliminary results indicate induction of human chondrocyte growth invitro, when incubated with chito-oligomers (unpublished data).

According to the hypothesis presented herein, the oligomer compositionsof the invention provide beneficial oligomeric substrates blockingchitinase-like proteins, as well as providing the known beneficialeffects of monomeric NAG and GlcN as oligomers are degraded.

It is further postulated herein that partially deacetylated polymericwater-soluble chitin will likewise provide anti-inflammatory effects anda therapeutic effect against joint disorders, as such water-solublepolymers allow some degradation by acidic chitinase and possiblychitolytic enzymes provided by the intestinal flora, providing in situwater-soluble chito-oligomers and NAG and glucosamine monomers.

In a first aspect, the invention provides a composition comprisingoligomers of N-acetyl glucosamine (NAG) and glucosamine, wherein atleast 50% of the chito-oligomers have a chain length in the range ofabout DP 2-50, and wherein the degree of deacetylation of the oligomersis in the range of about 0-70%.

In a further aspect a pharmaceutical composition is provided fortreatment of joint disorders in a subject in need thereof, whichcomposition is obtainable by partial deacetylation and partialdepolymerization of chitin.

Yet a further aspect of the invention provides the use ofchito-oligomers for manufacture of a medicament for treatment of jointdisorders, wherein the chito-oligomers comprise oligomers of N-acetylglucosamine (NAG) and glucosamine, wherein the chain length of thechito-oligomers is in the range of about 1-50, wherein at least 60 wt %of the chito-oligomers have a chain length of 2 or higher, and whereinthe degree of deacetylation of glucosamine is in the range of about0-50%.

The invention provides in another aspect the use of chito-oligomers formanufacture of a medicament for treatment against inflammatory activityin the human body.

LEGENDS TO FIGURES

FIG. 1. Biogel P4 GPC analysis of Sample 1. DP (polymer chain length)and homologues of each chain length are listed in Table 1.

FIG. 2. Homologue distribution of chito-oligomers in Sample 1 and Sample2 as determined by MALDI-TOF of whole samples. The relative signal fromthe analysis is calculated by adding the signals for all homologues fromDP2 to DP10 and adjusting to 100%. The signal for each homologue isexpressed as relative signal (%).

FIG. 3. Combined DP distribution of chito-oligomers from Sample 1 andSample 2, as judged by MALDI-TOF MS. Homologues for each DP, from DP 2to DP 10, as shown in FIG. 2, are added and expressed as combinedrelative signals (%).

DETAILED DESCRIPTION

The composition of the invention comprises oligomers of N-acetylglucosamine (NAG) and glucosamine, wherein at least 50% of thechito-oligomers have a chain length in the range of about 2-50, andwherein the degree of deacetylation of the oligomers is in the range ofabout 0-70% (F_(A)=0.3-1.0).

The term chito-oligomers as used herein refer to oligomers and polymersof one or both of N-acetyl glucosamine (NAG) and glucosamine, i.e.oligomer chains with a minimum chain length of 2 (dimers). As describedherein and in the examples set forth, the composition is particularlyuseful for use as a medicament.

The term homologue defines equal length oligomer chains with the sameratio of monomers, which may have different sequence, i.e., thehomologue A3D2 may comprise e.g., the oligomer sequences A-A-A-D-D andA-A-D-A-D. (A and D refer to N-acetyl glucosamine and glucosamine,respectively.)

Preferably the degree of deacetylation of the chito-oligomers is in therange of about 0-50%, and more preferably in the range of about 30-50%,and even more preferably about 35-50%, such as about 40-50%, includingabout 40% or about 50%. The degree of deacetylation DDA can also beexpressed as the acetylation factor, F_(A), where, e.g. DDA of 30%corresponds to F_(A)=0.7.

Preferably at least about 60% of the chito-oligomers have a chain lengthin the range of about 2-50, and more preferably at least about 75%, andyet more preferably at least about 85%.

The compositions of the invention may be suitably obtained fromchitinous raw material such as shrimp shells. Chitin is advantageouslydeacetylated with a strong base, such as by dissolving substantially drychitin in a concentrated base solution (e.g. 40-60% NaOH or KOH), at atemperature in the range of about 70-100° C. including the range ofabout 70-95° C., such as about 70-90° C., e.g. about 70° C., about 80°C., or about 90° C. The time of the reaction and concentration of chitinmay be varied depending on the desired degree of deacetylation, and canreadily be optimized for any particular processing unit and a particulardesired degree of deacetylation. The reaction is halted by washing theobtained chitin/chitosan with cold water, and the resulting solublechitin solution may be subjected to hydrolysis to obtainchito-oligomers, or the material may be dried with suitable drying meansfor subsequent further processing or storage.

As mentioned, enzymatic hydrolysis of the chitin/chitosan is preferredto obtain the chito-oligomers, however the use of suitable mineral acidsfor depolymerization (e.g. hydrochloric acid or nitrous acid) is alsoencompassed by the current invention. Several chitinase active enzymesare available and may be employed in this regard, e.g. chitinase (EC no.3.2.1.14) available from Sigma-Aldrich, also, lysozyme (EC no. 3.2.1.17)is found to have chitinase activity (see, e.g., U.S. Pat. No.5,262,310). The enzyme incubation conditions (enzyme/substrate ratio,temperature, pH, reaction time) may be varied, depending on the specificactivity and optimum reaction conditions of the employed enzyme. Asdemonstrated in Example 2 (see Sample 1 and 2; production), conditionsmay be optimized to obtain a desired ratio of small to medium-sizedoligomers. Longer oligomers and polymers (DP 30 and higher) mayoptionally be separated from the desired short and medium lengtholigomers, either by preparative chromatography, or by precipitation ata high pH (about pH 9 or higher).

The chito-oligomer composition may conveniently be provided in anessentially dry form comprising a powder, flakes or fibrous materialwhich can be capsulated or dissolved or suspended in an aqueous solutionfor intake. Such a composition may consist of substantially only theaforementioned oligomers, i.e. in the range of about 80-100 wt % of thechito-oligomers. In useful embodiments the composition comprises in therange of 20-100% by weight of said oligomers, including about 25-95 wt%, such as about 50-90 wt %. Depending on the manufacturing process, thecomposition may contain significant amounts of salt other than the saltsof oligomers, e.g. NaCl or KCl, but preferably the content of such extrasalts is kept to a minimum. Depending on the process and conditionsapplied during the hydrolysis of the raw material polymer, some amountof monomers of glucosamine and NAG are typically present in thecompositions of the invention, such as in the amount of 0-60 wt % of thetotal saccharide amount, such as less than about 50 wt %, but preferablythe monomers are less than about 40 wt % of the total saccharide amount,and such as less than about 25 wt %, including less than about 20 wt %.Our test results, however, indicate that a certain amount of monomers,in particular NAG, present in compositions of the invention may have apositive synergistic effect.

The composition may further comprise a pharmaceutically acceptableexcipient or diluent, a flavouring substance, a nutrient, or a colorant.

The shorter oligomers are postulated to be highly important for theactivity of the composition of the invention. In a useful embodiment atleast about 10 wt % of the oligomers of the composition have a chainlength of 2 to 12, more preferably at least 15 wt %, including at least25 wt %, and even more preferably at least 50 wt % of the oligomers havea chain length of 2 to 12. In a certain embodiment about 15 to 75 wt %of the oligomers have a chain length of 2 to 12, such as about 50 wt %of the oligomers, preferably about 15 to 75 wt % of the oligomers have achain length of 2 to 9. In certain embodiments at least 50 wt % of theoligomers have a chain length in the range of 2 to 15, such as at least60 wt %, including at least 70%, or at least about 80%.

In another aspect of the invention, a pharmaceutical composition isprovided, comprising the oligomer composition of the invention, asdescribed herein.

The pharmaceutical composition shall preferably be in a form suitablefor oral administration, such as a dry form which can be readilydissolved, e.g. in a glass of water. Such forms include dry powder,granular, flake, fibrous and paste forms. However, the composition canalso be contained in pills or capsules.

In other useful embodiments, the composition of the invention is in aform suitable for systemic administration, such as intramuscular,subcutaneous, or intravenous administration. Such suitable forms aresolution forms with a pharmaceutically acceptable carrier or excipientaccording to standard pharmaceutical practice. Said solution forms aresterile, and the pH is suitably adjusted and buffered. For intravenoususe, the total concentration of solute should be controlled to renderthe preparation isotonic.

As demonstrated in the accompanying examples, the pharmaceuticalcomposition is found to be useful for treatment of rheumatoid jointdisorders in a subject in need thereof, has been found particularlyuseful for the treatment of a joint disorder selected from the groupcontaining osteoarthritis and rheumatoid arthritis. In this context,treatment encompasses alleviating the symptoms of the joint disorder ina subject to whom the composition is administered.

In a further aspect, the invention provides a pharmaceutical compositionfor treatment of joint disorders in a subject in need thereof, whichcomposition is obtainable by partial deacetylation and partialdepolymerization of chitin. Such composition is preferably such asdescribed above.

In yet a further aspect, a composition is provided for anti-inflammatorytreatment and treatment of joint disorders comprising water-solublepartially deacetylated chitin with a degree of deacetylation in therange of about 35 to about 50%. The water-solubility of the polymerallows some degradation of the polymer by chitolytic enzymes produced bythe intestinal flora, thus the composition when ingested, provides insitu water-soluble chito-oligomers and glucosamine monomers, though tothe same extent as the above discussed hydrolysed chito-oligomers.

The invention provides in a further aspect a method of treating jointdisorders and inflammatory disorders comprising administering to asubject in need thereof a therapeutically active amount of oligomers ofN-acetyl glucosamine (NAG) and glucosamine, wherein the chain length ofthe chito-oligomers is in the range of about 2-50, and wherein thedegree of deacetylation of the oligomers is in the range of about 0-70%(F_(A)=0.3-1.0) and preferably in the range of about 0-50%, includingthe range of about 30-50%. In preferred embodiments, the methodcomprises administration of a composition such as described herein.

The joint disorders that may be advantageously treated with the methodof the invention include osteoarthritis and rheumatoid arthritis,inflammatory disorders, and other rheumatoid conditions.

In yet a further aspect, the invention provides the use of suchchito-oligomers as described above for the manufacture of a medicamentfor treatment of joint disorders, such as osteoarthritis and rheumatoidarthritis.

In another aspect, the use of the above-described compositions fortreatment against inflammatory activity in the human body is provided,as well as the use of said compositions for manufacture of a medicamentfor treatment against inflammatory activity, in bone and lean tissues.

EXAMPLES Example 1 Characterization of Chito-oligomers: AnalyticalMethods

1A: Determination of Water and Ash Content

A 4-5 g sample of spray-dried chito-oligomers was analysed for watercontent by gravimetric analysis before and after incubating at 105° C.for 3 hours. Ash content was determined by complete combustion at 800°C. for 3 hours and calculated as percent weight of inorganic residue ona dry weight basis.

1B: Determination of Degree of Deacetylation by Direct Titration

Chito-oligomers (500 mg, moisture and ash corrected) was mixed with 125ml 0.060 N HCl in a sealed Erlenmeyer flask and dissolved overnight at22° C. in a rotary shaker (150 rpm). Subsequently, 125 ml distilledwater was added and the solution was shaken further for at least 15 min.50.0 g of the solution were transferred to a beaker and titrated with0.500 N NaOH solution, using a flow of 1.00 ml/min (HPLC pump). The pHwas monitored between pH 1.8 to 9, and the DDA was calculated based onthe volume of the NaOH consumed between the inflexion points of thetitration curve, from pH 3.75 to pH 8.0, using the equation DDA=Vol (ml)NaOH* 16116*0.0500/100 mg chitosan. Each sample was titrated intriplicate.

1C: DNS Assay for Determining the Average Degree of Polymerisation (DP)

The average degree of polymerization (DP value) of the 0.50% oligomersolution was measured by a sugar reducing end assay using3,5-dinitrosalicylic acid (DNS) as a reagent and glucose as a standard.This method is originally described by Miller [34]. A volume of 1.00 mlof chitosan oligomer solution (5.00 mg/ml, moisture and ash corrected in0.5% acetic acid), was mixed with 2.00 ml of DNS reagent, boiled for 8min, cooled and centrifuged at 2000×g for 3 min. The optical density ofthe supernatant was measured in a spectrophotometer at 540 nm and theaverage DP-value was calculated using the absorbance of 1.00 mg/ml (5.55mM) glucose as a standard. Water (1.00 ml in 2.00 ml DNS solution)served as a blank at 540 nm. The average molecular weight used for DPcalculation was 200 Da. Each sample was assayed in duplicate.

1D: BioGel P4 Gel Permeation Chromatography Analysis (GPC)

Two serial columns (Pharmacia), with Biogel P4, fine grade (BioRad,Munchen, Germany). using 0.05 M ammonium acetate buffer adjusted with0.23 M acetic acid to pH 4.2 as mobile phase. The flow rate was 27.7ml/hr. Detection was done with Shimadzu RID 6A refractive indexdetector. Fractions were collected, appropriately combined, lyophilizedprior to MALDI-TOF MS analysis.

1E: MALDI-TOF Mass Spectrometry Analysis

Sample Preparation: Solutions of samples in H₂O (1 μL) were placed ontothe target and mixed with 1 μL of a 5% solution of THAP or DHB in MeOH.After drying at room temperature, the sample was re-dissolved in 1 μL ofMeOH to yield a thin layer of very fine crystals when dried at roomtemperature. Mass spectra were recorded with a Bruker Reflex IIInstrument (Bruker Daltonik, Bremen, Germany), as described in furtherdetail in [34].

Example 2 Production of Chito-oligomers (COs) Used for OralAdministration Against Arthritis.

Production of Sample 1 (G000823-1K):

Sodium hydroxide, 25 kg was dissolved in 25 kg of water in a 80 Lblender and heated to 70° C. Shrimp chitin (Primex ehf.), 2.5 kg wasadded and stirred (15 rpm) for 20 min. The slurry was then cooled withwater, filtered through a cheese-cloth bag (200×40 cm) and washed for10-15 minutes. The chitin gel was transferred back into the blender, thepH was adjusted to 4 by addition of 30% HCl, and water was added to givea volume of 80 L. Chitinase solution, 380 g (750 U/g) was added and thegel was stirred for 16 hrs at 30° C. The enzyme was denatured byadjusting the pH to 7 and heating of the solution to 70° C. for 10 min.After cooling, the oligomer solution was poured through a sieve of 280μm mesh size. The solution was subjected to spray-drying, using a rotaryatomizing spray-drying unit at an inlet air temperature of 190° C. andan outlet air temperature of 80° C. The atomizer rotor speed was 20,000rpm. The fine white chitosan powder, 2.0 kg was collected and kept atroom temperature, referred to as Sample 1.

Analysis of Sample 1

The spray-dried chito-oligomer sample was analysed for ash and watercontent. The ash content was 53.7% (w/w) and water 5.4% (w/w).Chito-oligomers and monomers were 40.9% (w/w). The degree ofdeacetylation (DDA) was 42.3%+/−0.1% (SD). Biogel P4 GPC (FIG. 1)followed by MALDI-TOF analysis (Table 1) showed the monomer (DP 1) beingmainly N-acetyl glucosamine (GlcNAc) with minor appearance ofN-glucosamine (GlcN). Dimers (DP 2) were a mixture of (GlcNAc)₂ and(GlcNAc)(GlcN). Trimers (DP 3) contained (GlcNAc)₂(GlcN) as main productand (GlcNAc)₃ as a minor product. The sequence of the main trimerproduct was determined to be GlcN-GlcNAc-GlcNAc or D-A-A. Longeroligomers (DP 4 to DP 20) were found in smaller quantity, as judged bythe Biogel P4 analysis. Existence of medium-length oligomers wasconfirmed by both Biogel P4 and MALDI-TOF MS analysis.

TABLE 1 MALDI-TOF MS of Biogel P4 GPC peaks from Sample 1 shown inFIG. 1. Each numbered peak was collected and analysed by MALDI-TOF MS.The table shows fraction number and calculated oligomers and homologuesof each fraction. 1 2 3 4 5 6 7 8 9 10 11 D8A4 D6A3 D4A3 D3A3 D2A3 D1A3D2A2 D1A2 A2 A2 A D7A5 D5A4 D3A4 D3A3 D3A2 D1A3 D1A2 D6A6 D4A5 D2A5 D4A2D2A3 D7A3 D6A4 D5A5 A = GlcNAc and D = GlcN.Production of Sample 2 (G010430-1K):

This sample was made essentially by the same protocol as described forSample 1, except after enzyme inactivation (pH adjusted to 8.0 by 10%NaOH) and sieving, the solution was clarified by using an Alfa-Lavalflow centrifuge (type: LAPX) at 9800 rpm. The liquid flow was 520ml/min. and the rotor was emptied every 3-5 min. The pellet wasdiscarded and the clear oligomer solution was spray-dried as in Example2. The product yield was 1.74 kg of powder.

Analysis of Sample 2

The ash content of this sample measured 48.3% (w/w) where NaCl contentwas 47.0%. Water content was 5.0% (w/w). Chito-oligomers and monomerswere 46.7% (w/w). The degree of deacetylation (DDA) was 38.7%+/−0.9.

MALDI-TOF MS analysis of Sample 1 and 2 are shown in FIGS. 2 and 3. FIG.2 indicates the homologue distribution of chito-oligomers of DP 2 to DP10. The homologue distribution is somewhat different between the twosamples. By adding the different homologues for the same DP it isevident that DP distribution is similar for the two samples, as shown inFIG. 3. It is important to keep in mind that the intensity signals fordifferent oligomers in the MALDI-TOF analysis are qualitative signals,not quantitative, and in particular that the peaks of higher oligomersmay appear with relatively lower intensities than the peaks of the loweroligomers.

Example 3 Oral Administration of Chito-oligomers (COs)

Subjects suffering from arthritis took daily doses of 3.0 g (1 tsp; 5.0ml, 1223 mg of COs) of Sample 1 spray-dried chito-oligomer powderdissolved in water for at least 5 weeks up to two years. Two of thesepatients stopped the administration for a 5-6 weeks period after acontinuous supplement and then started again taking 2.9 g of Sample 2 (1tsp; 5.0 ml, 1331 mg of COs).

Results of Administration

Subject 1: Treatment of Rheumatoid Arthritis

A female subject, age 55 years, was suffering from rheumatoid arthritis.The joints of both hands were severely swollen. The fingers were stiffand their movement caused pain. The subject took 3 g of the Sample 1chito-oligomer powder daily. The subject noticed a significantimprovement after 4 to 5 weeks. There was a remarkable relief ofsymptoms, inflammation ceased and joints of fingers appeared normalagain. There was a relief of pain and the subject could move her fingersmore freely, making her able to do delicate work again. In about 2months she stopped taking the chito-oligomer powder for 5-6 weeks. In 3to 4 weeks the arthritis symptoms gradually returned. Two to three weeksafter the cessation she started daily administration again, using 2.9 gof Sample 2 (1331 mg of COs), resulting in reported relief in 4 to 5weeks after the second onset of the administration. The subject has beenon a daily dose of Sample 1, 2 and similar chito-oligomer productionwithout inflammation and pain for about 21 month.

Subjects 2-4: Treatment of Rheumatoid Arthritis

The subjects were suffering from rheumatoid arthritis. They took 3.0 gof the Sample 2 chito-oligomer powder daily. After one month thesubjects reported significant relief of RA symptoms. Inflammation(swollen joints) was relieved and joints were less stiff.

Subjects 5-14: Treatment of Osteoarthritis

Ten subjects suffering from osteoarthritis took each 3.0 g daily ofSample 1, 2.9 g of Sample 2 and similar chito-oligomer production. After2 to 4 weeks, 8 subjects reported positive results, inflammation andpain was reduced. Two subjects reported no relief of symptoms.

For all subjects tested, no significant difference in relief of symptomswas found between Sample 1 and 2. Variations in sample preparation,different from Sample 1 and 2 (higher DDA, higher DP) have not led to animprovement in anti-arthritis activity, as judged by the subjects.Continuing trials are ongoing.

1. A method for treatment of a disorder selected from the groupcontaining joint disorders, inflammatory disorders, and other rheumatoidconditions, comprising administering a therapeutically effective amountof chito-oligomers of N-acetyl glucosamine (NAG) and glucosamineobtained by a process comprising a first step of partially deacetylatingchitin and a second step of enzymatically hydrolyzing the partiallydeacetylated chitin, wherein the oligomers have a chain length in therange of about 2-50, the degree of deacetylation of the oligomers is inthe range of about 0-70%, the oligomers comprise a size larger than adimer and have an inhibitory effect on chitinase-like proteins, and atleast about 10 wt % of the chito-oligomers have a chain length of 2 to12.
 2. The method of claim 1 wherein the degree of deacetylation ofchito-oligomers is in the range of about 30-50%.
 3. The method of claim1, wherein about 15 to 75 wt % of the chito-oligomers have a chainlength of 2 to
 12. 4. The method of claim 1, for treatment of a jointdisorder selected from the group containing osteoarthritis andrheumatoid arthritis.
 5. The method of claim 1, wherein the treatmentalleviates the symptoms of the joint disorder in a subject.
 6. Themethod of claim 1, comprising oral administration of saidchito-oligomers.
 7. The method of claim 1 wherein said step ofenzymatically hydrolyzing is performed using chitinase or lysozyme. 8.The method of claim 1 wherein said step of enzymatically hydrolyzing isperformed using lysozyme.